Abstract Genital malformation including hypospadias represents the second most common male birth defect after cardiac defect. In the past 50 years, hypospadias incidence has doubled along with other male reproductive problems. It is suspected that fetal exposure to endocrine disruptors may have contributed to this increase. However, the etiology of hypospadias is still largely unclear. Both environmental and genetic factors are involved. In fact, our understandings of genital development in general are still very limited. A complete understanding of genetic pathways governing genital development and masculinization and how perturbations of these pathways lead to genital malformations will have immense applications to improve global health. In the past few years, we have performed comprehensive genetic analyses on genital tubercle (GT) development in the mouse and established a conserved genetic pathway (Wnt/?-catenin?Sp8?Fgf8) in regulating body appendage outgrowth, including limbs and external genitalia. Genetic interactions between Wnt and Shh pathways in regulating both genitalia outgrowth and masculinization have also been described. Together, these studies laid the foundation for understanding posterior embryonic development as well as how environmental factors can influence genitalia development and cause hypospadias. Based on these findings, this proposal will continue to use mouse genetics including a series of conditional mutant mice to investigate a novel genetic pathway regulating GT outgrowth. In Aim I, We will characterize several knockout mouse models to build a genetic pathway regulating GT outgrowth and patterning. In Aim II, we will use a novel and highly innovative Split DamID technique to identify in vivo downstream targets of androgen receptor and ?-catenin during genital masculinization. Together, these studies should greatly improve our understanding of genitalia development and hypospadias formation. Our long term goal is to use mouse molecular genetics to understand the process of genital development and masculinization and the etiology of genital malformations, such as hypospadias.